Protein disulfide isomerase (PDI) is an enzyme involved in the catalysis of disulfide bond formation in secretory and cell-surface proteins. Using an oligonucleotide designed to detect the conserved "thioredoxin-like" active site of vertebrate PDI's (WCGHCK) (SEQ.ID.NO.: 1), we have isolated a gene encoding PDI from the lower eukaryote Saccharomyces cerevisiae. The nucleotide sequence and deduced open reading frame of the cloned gene predicts a 530 amino acid protein of molecular weight 59,082 and pI of 4.1, physical properties characteristic of mammalian PDIs. Furthermore, the amino acid sequence shows 30-32% identity and 53-56% similarity with mammalian and avian PDI sequences and has a very similar overall organization, namely the presence of two 100 residue segments, each of which is repeated, with the most significant homologies to mammalian and avian PDIs being in the regions (a, a') that contain the conserved "thioredoxin-like" active site. The N-terminal region has the characteristics of a cleavable secretory signal sequence and the C-terminal four amino acids (-HDEL)(SEQ.ID.NO.: 2) are consistent with the protein being a component of S. cerevisiae endoplasmic reticulum (E.R.). Transformants carrying multiple copies of this gene (designated PDI1) have 10-fold higher levels of PDI activity and overexpress a protein of the predicted molecular weight. The PDI1 gene is unique in the yeast genome and encodes a single 1.8 kb transcript that is not found in stationary phase cells, nor is it heat-inducible. Disruption of the PDI1 gene is haplo-lethal indicating that the product of this gene is essential for viability.
Protein disulfide-isomerase (PDI), an enzyme which catalyzes thiol:disulfide interchange reactions, is a major resident protein component of the E.R. lumen in secretory cells. A body of evidence on the enzymele cellular distribution, its subcellar location and its developmental properties suggests that it plays a role in secretory protein biosynthesis (Freedman, 1984, Trends Biochem. Sci. 9, pp.438-41) and this is supported by direct cross-linking studies in situ (Roth and Pierce, 1987, Biochemistry, 26, pp.4179-82). The finding that microsomal membranes deficient in PDI show a specific defect in cotranslational protein disulfide formation (Bulleid and Freedman, 1988, Nature, 335, pp.649-51) implies that the enzyme functions as a catalyst of native disulfide bond formation during the biosynthesis of secretory and cell surface proteins. This role is consistent with what is known of the enzyme's catalytic properties in vitro; it catalyzes thiol: disulfide interchange reactions leading to net protein disulfide formation, breakage or isomerization, and can catalyze protein folding and the formation of native disulfide bonds in a wide variety of reduced, unfolded protein substrates (Freedman et al., 1989, Biochem. Soc. Symp., 55, pp.167-192). The DNA and amino acid sequence of the enzyme is known for several species (Scherens, B. et al., 1991, Yeast, 7, pp. 185-193; Farquhar, R., et al., 1991, Gene, 108, pp. 81-89) and there is increasing information on the mechanism of action of the enzyme purified to homogeneity from mammalian liver (Creighton et al., 1980, J. Mol. Biol., 142, pp.43-62; Freedman et al., 1988, Biochem. Soc. Trans., 16, pp.96-9; Gilbert, 1989, Biochemistry 28, pp.7298-7305; Lundstrom and Holmgren, 1990, J. Biol. Chem., 265, pp.9114-9120; Hawkins and Freedman, 1990, Biochem. J., 275, pp.335-339). Of the many protein factors currently implicated as mediators of protein folding, assembly and translocation in the cell (Rothman, 1989, Cell 59, pp.591-601), PDI is unusual in having a well-defined catalytic activity.
PDI is readily isolated from mammalian tissues and the homogeneous enzyme is a homodimer (2.times.57 kD) with characteristically acidic pI (4.0-4.5) (Hillson et al., 1984, Methods Enzymol., 107, pp.281-292). The enzyme has also been purified from wheat and from the alga Chlamydomonas reinhardii (Kaska et al., 1990 Biochem. J. 268, pp.63-68). The activity has been detected in a wide variety of sources, and in a preliminary report, PDI activity was claimed to be detectable in S. cerevisiae (Williams et al., 1968, FEBS Letts., 2, pp.133-135). Recently, the complete amino acid sequences of a number of PDIs have been reported, largely derived from cloned cDNA sequences; these include the PDIs from rat (Edman et al., 1985, Nature, 317, pp.267-270) bovine (Yamauchi et al., 1987, Biochem. Biophys. Res. Comm., 146, pp.1485-1492) human (Pihlajaniemi et al., 1987, EMBO J., 6, pp.643-9), yeast (Scherens, B., et al., supra; Farquhar, R. et al., supra) and chick (Parkkonen et al., 1988, Biochem. J., 256, pp.1005-1011). The proteins from these vertebrate species show a high degree of sequence conservation throughout and all show several overall features first noted in the rat PDI sequence (Edman et al. 1985 supra). The most significant is the presence within the PDI sequence of two regions of approximately 100 residues strongly homologous to each other and closely related in sequence to thioredoxin, a small redox active-protein containing an active site disulfide/dithiol couple formed between vicinal Cys residues. In thioredoxin the active site sequence is WCGPCK (SEQ.ID.NO.: 3), whereas the corresponding region, found twice in PDI, has the sequence WCGHCK (SEQ.ID.NO.:1). (Other repeats, motif and homologies identified within the PDI sequences are discussed below).
Sequences corresponding to, or closely related to PDI have been identified in work aimed at analysing functions other than disulfide bond formation. For example, there is clear-cut evidence that PDI acts as the .beta. subunits of the tetrameric .alpha..sub.2.beta..sub.2 enzyme prolyl-4-hydroxylase, which catalyzes a major post-translational modification of nascent or newly-synthesized procollagen polypeptides within the E.R. (Pihlajaniemi et al., 1987, supra; Koivu et al., 1987, J. Biol. Chem., 262, pp.6447-49)). There is also evidence suggesting that PDI participates in the system for cotranslational N-glycosylation (Geetha-Habib et al., 1988, Cell, 4, pp.63-68) and recently the proposal has been made that the enzyme participates in the complex which transfers triglyceride to nascent secretory lipoproteins (Wetterau at al., 1990, J. Biol. Chem., 265, pp.9800-7). Thus, PDI may be multifunctional in the co- and post-translational modification of secretory proteins (Freedman, 1989, Cell, 57, pp.1069-72).
The vast majority of mammalian secretory proteins contain multiple intramolecular and/or intermolecular disulfide bonds. Examples include, but are not limited to, pituitary hormones, interleukins, immunoglobulins, proteases and their inhibitors and other serum proteins. Such proteins are among the prime targets for commercial genetic engineering, but early experience in their expression in bacteria and yeast has highlighted a number of problems in obtaining them as functionally active recombinant products. This has drawn attention to the need for a better understanding of post-translational modifications in general, and of protein folding and disulfide bond formation in particular.
Disulfide bonded proteins comprising a single folded domain can, in general, be fully reduced and denatured and subsequently renatured in vitro to generate the correctly disulfide-linked state in reasonable yield. The process involves rapid formation of a mixed population of many differently disulfide bonded forms which slowly isomerize to give the native disulfide pairing. The process is catalysed by thiol/disulfide redox buffers (e.g. GSH and GSSG) and by alkaline pH. Low protein concentrations are required to prevent precipitation and interchain disulfide formation. In general the rate of formation of the native protein, and the optimal obtainable yield, both decrease as the number of intramolecular disulfides increases. The problem is compounded in proteins containing multiple disulfide bonded domains (e.g. tissue plasminogen activator) in which each domain must fold and form its native disulfide bonds independently.
The process of disulfide bond formation in vivo occurs co-translationally or as a very early post-translational event. Studies on nascent and newly synthesized secretory proteins in the lumen of the E.R. in mammalian cells show that native disulfide bonds are already formed. The process in vivo appears to be catalyzed by the enzyme protein disulfide-isomerase which is an abundant protein in secretory cells and is located at the luminal face of the endoplasmic reticulum [Freedman, R. B., 1984, Trends in Biochemical Sciences, 9, 438-441]. This enzyme in vitro, catalyzes thiol:protein-disulfide interchange reactions in a wide range of protein substrates and has the properties required of a cellular catalyst of native protein disulfide formation [Freedman, R. B. et al., 1984, Biochem. Soc. Trans., 12, 939-942]. Further evidence for its role include (i) that its tissue distribution matches that of the synthesis of disulfide bonded secretory proteins [Brockway, B. E. et al., 1980, Biochem, J., 191, 873-876], and (ii) that in a number of systems the amount of enzyme present varies in parallel with a physiological change in the rate of synthesis of disulfide bonded secretory protein [Brockway, B. E. et al., 1980, Biochem J., 191, 873-876; Freedman R. B. et al., 1983, in "Functions of Glutathione: Biochemical, Physiological, Toxicological & Clinical Aspects". eds. A. Larsson, S. Orrenius, A. Holmgren & B. Mannervik, Raven Press, New York, pp.271-282; Paver, J. L. et al., 1989, FEBS Letters, 242, pp. 357-362].
The enzyme has been characterized in a number of animal sources [Lambert, N. and Freedman, R. B., 1983, Biochem. J., 213, pp. 225-234], and in wheat [de Azevedo, G. M. V. et al., 1983, Biochem. Soc. Trans., 12, 1043], and a striking conservation of molecular and kinetic properties has been noted [Freedman, R. B. et al., 1984, Biochem. Soc. Trans. 12, pp. 939-942; Brockway, B. E. and Freedman, R. B., 1984, Biochem J., 219, 51-59]. However the enzyme has not been throughly studied in lower eukaryotes or in bacteria. The strong homologies between yeast and higher eukaryotes in the mechanisms and molecular components involved in secretion strongly suggest that the enzyme or an analogue is present in yeast, since at least some yeast secretory proteins (e.g. killer toxin) contain disulfide bonds.
The application of yeast as a versatile host for the expression of commercially-important mammalian proteins is compromised, to some extent, by the limited capacity of the yeast secretory system and by some differences between it and that of higher eukaryotes (e.g. in glycosylation).
The present invention provides a novel process for the production of disulfide bonded proteins in a recombinant host cell overexpressing the enzyme protein disulfide isomerase, and provides recombinant yeast cells which overexpress protein disulfide isomerase. The present invention also provides recombinant yeast host cells which substantially and unexpectedly increase the secretion of a recombinant disulfide bonded, secreted protein.